This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. In 2009, we started a new project: isolation of bacteria from nearby alkaline lakes. The project has involved the isolation and identification of alkaliphilic bacteria. These bacteria may be potentially useful in industry or in the study of adaptations to extreme environments. Several bacteria have been isolated and grown, and we have selected a few that are true alkaliphiles for further study. Within the group, Katie Score is focusing on identification of non-fastidious bacteria and on techniques to study populations of bacteria. Katie Brown (who graduated in December) and now Luke Wright (a non-INBRE student) are working on culture media and techniques to isolate more fastidious bacteria. Both groups have had some success, and we are working on preparing more DNA for sequencing to identify the rest of the alkaliphiles we have isolated. Here is the abstract submitted to the Nebraska Academy of Sciences. Southwestern Nebraska is dotted with hundreds of alkaline lakes, ranging from pH 9-11. The bacteria that grow in these lakes have adapted to the alkaline conditions and are alkalitolerant (able to live in both alkaline and more neutral environments) or alkaliphilic (adapted to live only in alkaline environments). The purpose of this study was to identify alkaliphilic bacteria and to eventually study the adaptations these bacteria have made in order to survive in an alkaline environment. Water was collected from several ponds and lakes along Highway 2 near Antioch, NE. The pH value of these bodies of water ranged from pH 9-11. Bacteria were then grown on plates of defined media at pH 7, pH 9, and pH 11. Bacteria that could grow at pH 9 or pH 11 but not at pH 7 were considered to be alkaliphilic. The genomic DNA from these bacteria was harvested and amplified in PCR reactions using the universal bacterial primers for 16S rDNA. This amplified DNA was then sequenced in an attempt to identify the alkaliphilic bacteria. Tentatively identified bacteria include Bacillus pseudofirmus, an alkaliphilic and halotolerant bacteria, and a potentially new species of Exiguobacterium, a genus that includes both psychrophilic and thermophilic bacteria. These bacteria will be sequenced and studied to determine how they adapt to their alkaline environment.